DNA Extraction for college laboratory setting
Label one tube for each plant .
Harvest 2 - 3 seedlings and place in a mortar .
Fill with about 50 ml of liquid nitrogen .
Grind tissue with pestle .
Add 1 ml of extraction buffer to the tube .
Add 120 µl of 10 % SDS .
Mix by inverting .
Incubate tube ( s ) at 65 ˚C for 20 minutes .
Add 300 µl 5M KOAc .
Mix well by inverting several times ( important ! ) , then place on ice 5 minutes .
Centrifuge for 5 minutes at > 12,000 rpm .
Label a second tube .
Pass 700 µl of the supernatant through a miracloth funnel into the second tube .
Add 600 µl of isopropanol .
Mix the contents thoroughly by inverting .
Spin for 5 minutes at 14,000 rpm .
Carefully pour off and discard the supernatant .
Use a P20 set to 20 µl to remove the remaining drops of liquid without disturbing the DNA pellet .
Add 500 µl of 70 % ethanol and flick the tube until the pellet comes off the bottom .
Spin 5 minutes .
Pour off the ethanol .
Use a P20 set to 20 µl to remove the remaining drops without disturbing the pellet .
Leave the tube open on the bench to air dry for 5 - 10 minutes .
Resuspend the DNA in 50 µl TE and incubate at room temperature for 5 minutes for complete resuspension .
Samples should be frozen for storage .
